High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase

Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes.     

Molecular cloning of the mouse 46-kDa mannose 6-phosphate receptor (MPR 46).

A cDNA clone for the mouse 46-kDa mannose 6-phosphate receptor (MPR 46) was isolated from an embryonic mouse cDNA library. Its single open reading frame codes for a protein of 278 residues. It shows an over-all amino-acid identity of 93% with the human receptor. Nine non-conservative amino-acid exchanges are found in the luminal domain, one non-conservative exchange of hydrophobic amino acids is in the transmembrane domain, while the cytoplasmic receptor tails are identical. All five potential N-glycosylation sites are conserved as well as amino acids that are important for ligand binding (Arg 137 and His 131) and disulfide pairing (Cys 32 and 78, Cys 132 and Cys 167, Cys 145 and Cys 179). The absolute identity in the cytoplasmic MPR 46 tail suggests the importance of this amino-acid sequence for the intracellular routing of the MPR 46.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46.     

Stimulated and co-operative electron transfer in energy conversion and catalysis.

A new mechanism of electron transfer, stimulated electron transfer, is postulated, in which an electronic feedback is drastically increasing both the rate of electron transfer and the propagation of free energy along electron transferring molecular pathways. In principle, the idea of pushing a system far from equilibrium to achieve a high reaction rate and co-operative phenomena is applied to molecular electron transfer. The effect is calculated from a semiclassical kinetic model of a chain redox reaction with autocatalytic feedback on individual rate constants, where the steps have subsequently been minimized to obtain a continuous electron transfer pathway with electronic feedback. The influence of inhomogeneities and asymmetries in the electron transfer path and of vectorial components (electrical field, gradient of redox potential) are discussed as well as the acceleration of individual and multiple electron transfer as a function of feedback. Examples of autocatalytic feedback are provided including mechanisms involving electron transfer proteins and multi-centre electron transfer catalysts. Such a phenomenon can be described for molecular and interfacial electron transfer in analogy to stimulated and coherent light emission. The results suggest that autocatalytic or stimulated electron transfer may be a key to the understanding of efficient electron transfer and co-operative multi-electron transfer catalysis in biology and a challenge for fuel production mechanisms in artificial photosynthesis and fuel cycles.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Die cucurbitacine in Bryonia alba und Bryonia dioica

The cucurbitacins in roots of Bryonia dioica and B. alba have been investigated. Both species contain the cucurbitacins E, B, I, D, J, K and L, the dihydrocucurbitacins E and B, and tetrahydrocucurbitacin I. The detection of certain cucurbitacin aglycones depends upon the date of harvest, the duration of storage and the methods used for extraction.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1

Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.     

Enlargement of Cerebral Ventricles as an Early Indicator of Encephalomyelitis

Inflammatory disorders of the central nervous system such as multiple sclerosis and acute disseminated encephalomyelitis involve an invasion of immune cells that ultimately leads to white matter demyelination, neurodegeneration and development of neurological symptoms. A clinical diagnosis is often made when neurodegenerative processes are already ongoing. In an attempt to seek early indicators of disease, we studied the temporal and spatial distribution of brain modifications in experimental autoimmune encephalomyelitis (EAE). In a thorough magnetic resonance imaging study performed with EAE mice, we observed significant enlargement of the ventricles prior to disease clinical manifestation and an increase in free water content within the cerebrospinal fluid as demonstrated by changes in T₂ relaxation times. The increase in ventricle size was seen in the lateral, third and fourth ventricles. In some EAE mice the ventricle size started returning to normal values during disease remission. In parallel to this macroscopic phenomenon, we studied the temporal evolution of microscopic lesions commonly observed in the cerebellum also starting prior to disease onset. Our data suggest that changes in ventricle size during the early stages of brain inflammation could be an early indicator of the events preceding neurological disease and warrant further exploration in preclinical and clinical studies.